Microbiological Research 229 (2019) 126328

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Microbiological Research

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Diversity of cultivable endophytic in mulberry and their potential for antimicrobial and plant growth-promoting activities T

Weifang Xua,b, Fei Wanga,b, Meng Zhanga,b, Ting Oua,b, Ruolin Wanga,b, Gary Strobelc, ⁎ ⁎ Zhonghuai Xianga,b, Zeyang Zhoua,b,d, , Jie Xiea,b, a State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University, Chongqing, 400715, China b Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture, College of Biotechnology, Southwest University, Chongqing, 400715, China c Department of Plant Sciences, Montana State University, Bozeman, MT, 59717, USA d College of Life Science, Chongqing Normal University, Chongqing, 400047, China

ARTICLE INFO ABSTRACT

Keywords: Endophytic bacteria-based biocontrol is regarded as a potential plant disease management strategy. Present Endophytic bacteria study analyzed the diversity of mulberry endophytic bacteria basing on a culture-dependent approach and Diversity further evaluated their antimicrobial and plant growth-promoting (PGP) activities. A total of 608 cultivable Mulberry fruit sclerotiniosis endophytic bacteria, belonging to 4 phyla and 36 genera, were isolated from four mulberry cultivars having Biological control different resistance to sclerotiniosis in three seasons. Taxonomic compositional analysis results showed that Plant growth promotion , Firmicutes, and were the three dominant bacterial phyla in all communities, with the representative genera Pantoea, Bacillus, , Curtobacterium, and Sphingomonas. Diversity analysis results indicated that the diversity of winter community was higher than that of spring or autumn, and higher diversities were detected in the resistant cultivar communities compared with the susceptible cultivar. Antagonism assays results showed that 33 isolates exhibited strong and stable activity against three phyto- pathogens which are Sclerotinia sclerotiorum, Botrytis cinerea, and Colletotrichum gloeosporioide. Eight endophytic bacteria were selected out from 33 antagonists based on the evaluation of antagonistic and PGP activities. Furthermore, pot experiment results revealed that all the 8 tested endophytes stimulated the growth of mulberry seedlings at different levels, and Bacillus sp. CW16-5 exhibited the highest promotion capacity, which the shoot length and the root fresh weight were increased by 83.37% and 217.70%, respectively. Altogether, present study revealed that mulberry harbors a large amount of diverse cultivable endophytic bacteria and they also serve as novel sources of beneficial bacteria and bioactive metabolites.

1. Introduction Wolf, 1945; Sultana et al., 2013), Sclerotinia sclerotiorum (Lü et al., 2013), and Scleromitrula shiraiana (Hong et al., 2007), can infect mul- Mulberry (Morus L.), as the irreplaceable food of silkworm (Bombyx berry flowers resulting in the occurrence of diseased mulberry fruit. To mori), played a very important role in the Silk Trade of ancient China. date, chemical and nonchemical managements have been used to Mulberry fruit, as the byproduct of sericulture in the past, has been control mulberry fruit sclerotiniosis. Effective chemical prevention proved to contain abundant vitamins, minerals, and bioactive sub- methods are currently being used worldwide to control this disease (Lü stances that are beneficial for human health (Liang et al., 2011; Sultana et al., 2011; Ye et al., 2014; Ju et al., 2016), but long-term overuse of and Kim, 2016). In recent years, the planting area of the mulberry tree chemical fungicide is undoubtedly a threat to human health, with in- has been remarkably increased due to the high demand for mulberry creasing environmental pollution and inducing the emergence of drug- fruit for human use (Kuai and Wu, 2012; Sultana et al., 2013). How- resistant pathogens (Commare et al., 2002). Moreover, other ap- ever, mulberry fruit productivity is greatly threatened by the white fruit proaches, such as breeding for resistance and traditional cultural con- disease, a soil-borne fungal disease called ‘mulberry fruit sclerotiniosis’. trol practices (rotation, soil management, and nonwoven fabric Four species in the family Sclerotiniaceae, namely Ciboria shiraiana mulching) have failed to show significant effects against this fruit dis- (Hong et al., 2007; Hu et al., 2011), Ciboria carunculoides (Whetzel and ease (Bae et al., 2010). In recent years, microbe-based biological control

⁎ Corresponding authors at: State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University, Chongqing 400715, China. E-mail addresses: [email protected] (Z. Zhou), [email protected] (J. Xie). https://doi.org/10.1016/j.micres.2019.126328 Received 18 April 2019; Received in revised form 3 August 2019; Accepted 30 August 2019 Available online 31 August 2019 0944-5013/ © 2019 Elsevier GmbH. All rights reserved. W. Xu, et al. Microbiological Research 229 (2019) 126328 has been considered as a potential and sustainable alternative for 2. Materials and methods combatting phytopathogens and promoting plant growth. Endophytes are often defined as nonpathogenic bacteria and fungi 2.1. Mulberry sample preparation that reside in the living tissue of healthy plants without causing ap- parent harm to their hosts (Bacon and White, 2000; Carroll, 1988). Healthy branches from four healthy mulberry cultivars with dif- Endophytic bacteria, as important components of the plant microbiota, ferent degrees of resistance to sclerotiniosis (Changguo Sang = CGS, have been isolated from diverse plant species, and some have exhibited Chuan Sang No.7637 = CSQ, Xin Lunjiao = XLJ, and Hong Guo considerable potential for the application of plant protection areas. No.2 = HGE) were collected in this study: cultivars CGS and CSQ were Some of these bacteria can accelerate seed germination and promote resistant to sclerotiniosis, and cultivars XLJ and HGE were susceptible plant growth by several mechanisms, including nitrogen fixation, to the sclerotial disease (Huang et al., 2012). Among the four cultivars, phosphate solubilization, siderophore production, and bioactive en- CGS was collected at the Southwest University experimental farm (29° zyme secretion (Liotti et al., 2018; Pereira et al., 2016). Some large 49ʹ 1ʹʹ N, 106° 24ʹ 57ʹʹ E), and the remaining three were obtained from populations of endophytic Bacillus (Zouari et al., 2016), Pseudomonas the Sericulture Science and Technology Institute experimental farm (Wicaksono et al., 2018), and Pantoea (Xie et al., 2017) species dis- (29° 50ʹ 39ʹʹ N, 106° 25ʹ 55ʹʹ E). Both farms were located in an area near played strong antagonistic activity against phytopathogenic fungi by the north side of the Jialing River in Chongqing, China, and the two producing a variety of secondary metabolites with antimicrobial regions share same climate conditions. Two-year-old stems with ap- properties. In addition, part of endophytes showed good colonization proximately 1.5∼2.0 cm in diameter were collected in January, April, ability of plant tissue, and this capacity can help them to confer bene- and September, representing the seasons winter, spring, and autumn, ficial effects on host growth, because colonization behavior is believed respectively, for the two consecutive years 2015∼2016. After removing as a critical process for the interaction between beneficial microbes and leaves and small branches, the samples were immediately transported plants (Compant et al., 2010; Sharma et al., 2008). Furthermore, ana- back to the laboratory and stored at 4 °C until further processing. logous to the beneficial microbes in different environments, endophytic microorganisms also act as reservoirs of novel bioactive substances 2.2. Isolation of endophytes (Strobel, 2003) and thus have applications in medicine, agriculture, and industry. Due to their myriad of functions, endophytic bacteria can be Surface sterilization of the mulberry stems was performed according regarded as good candidates for natural biological control antagonists to a previously described procedure (Strobel et al., 2000), and the en- (BCAs). dophytic bacteria were isolated using the fragmentation technique In fact, management of such biological control programs, using (Liotti et al., 2018). In brief, samples were washed with tap water to endophytic bacteria as BCAs, requires some understanding of the remove soil and other debris before being cut into pieces with structure and dynamics of microbial endophytic communities (Liotti 3.5∼5.0 cm in length. The samples were then thoroughly soaked in a et al., 2018). Numerous studies have demonstrated that plant species, 70% ethanol/water (v/v) solution and rapidly flame-sterilized. Then, cultivars, and some environmental factors are key determinants of en- the stems were peeled to obtain smaller fragments and placed on water dophytic composition and function. Shen and Fulthorpe (2015) in- agar (WA), Gause's agar (GA), and potato dextrose agar (PDA) medium, dicated that the diversity of endophytic bacteria in urban trees (Acer respectively. The plates were incubated at 28 °C for 20 days and ex- negundo, Ulmus pumila, and Ulmus parvifolia) was highly dependent on amined daily for the presence of colonies at the edge of the stem piece. the season. Mocali et al. (2003) found that bacterial communities as- Colonies with different morphological characteristics were chosen from sociated with elm were influenced by variations in seasons and organs. each plate and purified using a streak plate on PDA medium. All the Additionally, significant differences in the densities and structures of purified isolates were stored with 30% glycerol at −80 °C. endophytic microbial communities have also been detected in different cultivars or genotypes of plants (da Silva et al., 2014; Liotti et al., 2018; 2.3. Classification of endophytic bacteria van Overbeek and van Elsas, 2008). Studies on the determinants of endophytic community structure have been reported for many crops, Classification of the bacteria was based on the analysis of 16S rRNA such as maize (da Silva et al., 2014), cotton (Adams and Kloepper, gene sequencing using the universal primers 27 F/1492R (Bredow 2002), and potato (Marques et al., 2015), but information on the en- et al., 2015). The total DNA of the strain was extracted with PrepMan dophytic bacterial community of mulberry remains scarce. Ultra Sample Preparation Reagent kit (Applied Biosystems, Palo Alto, In recent decades, some endophytic strains isolated from mulberry CA, USA) according to the manufacturer’s instructions. The DNA from have exhibited strong antagonistic activity against various phyto- all the purified isolates was used for PCR amplification of the 16S rRNA pathogens (Mu et al., 2008; Tan et al., 2012; Xie et al., 2017), but very gene, and this reaction was carried out in a 25-μL volume with the few isolates have been used to effectively control the sclerotial disease following conditions: one cycle of 95 °C for 4 min; followed by 30 cycles of mulberry fruits. The aims of the present study were to (i) isolate of 94 °C for 30 s, 50 °C for 45 s, and 72 °C for 1 min; and a final extension cultivable endophytic bacteria and analyze the diversity of these com- at 72 °C for 8 min. The PCR-amplified products were purified with the munities from four mulberry varieties during winter, spring, and au- DNA Clean & Concentrator™-5 Kit (Zymo Research, USA) and then se- tumn in two consecutive years (2015∼2016); (ii) screen for endophytic quenced by the Sanger method at Sangon Biotechnology Co., Ltd., bacteria with antagonistic activity against S. sclerotiorum in vitro and Shanghai, China. The generated sequences were aligned using BioEdit further evaluate the inhibition activity of some antagonists toward software version 7.0 and then subjected to analysis by the Basic Local other phytopathogens and their capability with production of plant Alignment Search Tool (BLAST) search program of the NCBI database growth-promoting (PGP) traits; and (iii) assess the effects of endophytes (National Center for Biotechnology Information, https://blast.ncbi.nlm. with potential biological control applications on the growth of mul- nih.gov/Blast.cgi) to determine the sequence homology with closely berry seedlings under greenhouse conditions. Ultimately, the goal of related organisms (Altschul et al., 1997). In present study, the micro- present research was to establish a microbial collection of mulberry organisms with high level of identity (97–100%) were selected as the endophytes and further obtain resources of bioactive endophytes with closest match, and all bacterial isolates were respectively classified to potential applications in the biological control of mulberry fruit scler- the level according to the information of the closest microbes. In otiniosis. addition, the taxonomic database of NCBI was used to classify all the endophytic bacterial strains, including the level of phylum, class, order, and family. All 16S rRNA gene sequences obtained in this study were submitted to GenBank under the accession numbers MH768978-

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MH769584 and MF375212 (Fang et al., 2018). days. Plates that were inoculated with a pathogen plug of the same size were tested in the absence of endophytic bacterial cells as a control. The 2.4. Endophytic bacterial community analysis inhibition rate (I) was calculated using the following formula: I(%)=

[(C–T)/(C-C0)]×100, where C represented the growth diameter of the To understand the impacts of seasons and mulberry cultivars on the fungal pathogen in the control, T represented the growth diameter of distribution of endophytic bacteria, 7 endophytic bacterial commu- the pathogen in the dual culture plate, and C0 represented the diameter nities were constructed. Three communities were based on seasons: (1) of the test fungal agar discs (5.0 mm). All experiments were carried out winter, community isolated in the winters of 2015 and 2016; (2) spring, in triplicate. community isolated in the springs of 2015 and 2016; and (3) autumn, community isolated in the autumns of 2015 and 2016. In addition, four communities were based on mulberry cultivars: (1) CSQ, community 2.5.2. PCR detection of genes related to antibiotic biosynthesis isolated from cultivar CSQ; (2) CGS, community isolated from cultivar Direct antagonism of phytopathogens is a key biocontrol mechanism ffi CGS; (3) XLJ, community isolated from cultivar XLJ; and (4) HGE, for most BCAs, and this mechanism is dependent on e cient antibiotics community isolated from cultivar HGE. production, mainly secondary metabolites with antimicrobial activity. The diversity of different endophytic bacterial communities was Among these compounds, polyketides and lipopeptides were the two compared at genus level using the following indices (Qadri et al., 2014): major representatives, especially nonribosomally synthesized cyclic li- 1) Isolation frequency (IF), which was calculated as the number of popetides (surfactin, iturin, and fengycin) (Cawoy et al., 2015). To in- isolates from a certain genus divided by the total number of isolates vestigate the putative antifungal mechanism of the antagonists, the from all samples; 2) Simpson’s diversity index (D), which was calcu- functional genes associated with biosynthesis of antimicrobial sub- lated as D =1−Ʃpi2, where pi=Ni/N, Ni was the number of isolates of stances were determined by a PCR assay (Gond et al., 2015; Wang et al., the genus i and N was the total number of isolates from a certain 2016). Primers were synthesized according to sequences chosen from community; 3) Shannon-Weaver diversity index (H′), which was cal- the coding regions of PKSI (polyketide synthase), NRPS (nonribosomal culated as H′=−Ʃpi (ln pi); and 4) Pielou’s evenness (E), which was peptide synthetase), sfp (surfactin biosynthesis), srfC (surfactin syn- calculated as E=H′/Hmax, where Hmax=ln (S), with S as the total thase), ItuD (iturin A biosynthesis), and FenD (fengycin biosynthesis). fi number of genera in a certain community. In addition, Venn diagrams The six primers used for ampli cation of the functional genes are listed (http://bioinformatics.psb.ugent.be/webtools/Venn/) were con- in Table S1. structed for all classified genera (Liotti et al., 2018). 2.5.3. Determination of plant growth-promoting (PGP) traits of antagonists 2.5. Screening of mulberry endophytic bacteria as potential biocontrol The PGP traits of the antagonists, including phosphate solubiliza- agents controlling mulberry fruit sclerotiniosis tion, siderophore production, nitrogen fixation, and hydrolytic enzymes activity (cellulose, protease, and chitinase) were qualitatively de- The following procedure for screening the bioactive endophytes was termined by following standard procedures. A cell suspension of each summarized in Fig. S1 (Liotti et al., 2018; Passari et al., 2016). antagonist was prepared as described above and 10 μL of each culture was spotted on different agar medium plates, respectively. Pikovskaya’s 2.5.1. Determination of antimicrobial activity of endophytic bacteria (PVK) agar medium containing tricalcium phosphate (Ca3[PO4]2)(Vyas S. sclerotiorum, the fungal pathogen that causes mulberry fruit et al., 2007), chrome azurol-s (CAS) agar medium (Jasim et al., 2013), sclerotiniosis, was used as a target to assay the biocontrol potential of and nitrogen-free (NFM) agar medium (Ben Abdallah et al., 2018) was fi all the endophytic isolates. Each puri ed endophytic bacteria was used for evaluation of phosphate solubilization, siderophore produc- grown on a LB plate at 30 °C for 24 h and one colony for each strain was tion, and nitrogen fixation, respectively. The cellulose, protease, and inoculated into LB medium and incubated at 30 °C for 96 h at 180 rpm. chitinase activities was detected on carboxymethyl cellulose (CMC) The cultures were then centrifuged at 10,000 × g for 30 min to discard agar medium containing 0.2% (w/v) Congo red, skim milk agar (SMA) the cells, and the cell-free supernatant was obtained by passing the medium, and half strength tryptic soya agar (TSA) medium supple- μ fi supernatant through a 0.22- m ltration membrane. The antagonistic mented with 0.6% (w/v) colloidal chitin, respectively (Afzal et al., activity of all the endophytic bacteria was qualitatively assayed by the 2017; Patagundi et al., 2014). These activities were qualitatively eval- ff well di usion technique (Zouari et al., 2016). In brief, S. sclerotiorum uated by the presence of a transparent zone or hydrolysis-induced halo agar discs (5.0 mm) were placed at the centers of PDA plates, and around the bacterial colony after 7 days of incubation at 30 °C, whereas μ 100 L of cell-free supernatant was added into wells that were 3.0 cm nitrogen fixation activity was indicated by positive growth on the from the center. The plates were incubated at 25 °C for 5 days, and the medium after 2 days of incubation at 30 °C. All experiments were per- diameters (Di) of the fungal inhibition zones were measured. Only formed in triplicate. freshly transferred cultures were used for all the tests, and all treat- ments were performed in triplicate. To make antagonistic strains wider application in biological control 2.5.4. Effects of the antagonistic endophytes on the growth of mulberry of common plant diseases, the isolates that exhibited strong antag- seedlings onistic activity against S. sclerotiorum in a qualitative bioassay were The antagonistic isolates, exhibiting good antifungal ability and selected for further quantitative testing of their activity against S. high PGP potential, were selected from the dominant groups of mul- sclerotiorum and two other fungal pathogens Botrytis cinerea and berry endophytic bacteria and subjected to further evaluation of their Colletotrichum gloeosporioide, of which the former can cause gray mold effects on mulberry seedling growth. Ten milliliters of bacterial culture, of multiple fruits (Jurick et al., 2017; Williamson et al., 2007) and the which was prepared in King’s medium (Glickmann and Dessaux, 1995) latter can infect a wide range of plant species by a specialized infection and adjusted to a density of 1.0 × 107 CFU/mL with sterilized distilled structure called appressorium (Huang et al., 2016; Priyatno et al., water, was applied to each pot in which 25-day mulberry seedlings 2012). The antifungal bioactivities were evaluated on PDA medium by were planted. Seedlings inoculated with water served as a control. Five the dual culture technique (Passari et al., 2016). Briefly, a 5-mm pa- pots with three plants per pot were used in each treatment. After in- thogenic mycelial disc was placed in the center of a PDA plate, and then oculation forty-five days, five seedlings were randomly selected from the tested bacterial culture in logarithmic phase (108 colony forming each treatment to measure parameters associated with plant growth, units per microliter, (CFU/mL)) was streaked on opposite sides of the including the root and shoot length, and fresh weight of root and shoot same plate with 3.0 cm from the center and incubated at 25 °C for 5 (Xie et al., 2017).

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Table 1 the CGS community, 143 from the HGE community, and 149 from the Diversity profile of the endophytic bacterial communities in mulberry. XLJ community) (Table 1). Based on the 16S rRNA gene sequencing

Seasons Mulberry cultivars results, culturable bacterial isolates were divided into 36 genera, which were distributed into 4 phyla, 7 classes, 13 orders, and 20 families as winter spring autumn CSQ CGS HGE XLJ shown in Table 2. All of the isolates belonged to the phyla Proteobacteria, Firmicutes, Number of isolates 127 188 293 147 169 143 149 fi Number of genera 18 22 23 23 22 15 13 Actinobacteria, and Bacteroidetes, and the rst three were the pre- Shannon-Weaver (H’) 2.18 2.04 1.75 2.21 2.04 1.67 1.82 dominant phyla among the endophytic bacterial communities of mul- Simpson’s index (D) 0.83 0.81 0.72 0.84 0.78 0.70 0.78 berry (Table 2). Under the most abundant phylum Proteobacteria (382 Pielou’s evenness (E) 0.75 0.66 0.56 0.71 0.66 0.62 0.71 of the 608 isolates, accounting for 62.83%), bacteria belonging to the classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteo- Diversity indices were calculated at the genus level. Winter, spring, and autumn bacteria were detected, and a majority of the isolates were obtained represent the community isolated in the winters, springs, and autumns of 2015 and 2016, respectively; CSQ, CGS, HGE, and XLJ represent community isolated from the class (337 of the 382 isolates, ac- from Chuan Sang No. 7637, Changguo Sang, Hong Guo No. 2, and Xin Lunjiao, counting for 88.22%). As the second most dominant bacterial phylum, respectively. Firmicutes only contained the class Bacilli (163 isolates), consisting of Bacillus spp. (158 of the 163 isolates, accounting for 96.93%) and 2.5.5. Statistical analysis Paenibacillus spp. (5 of the 163 isolates, accounting for 3.07%). Isolates Data for bacterial promoting plant growth were analyzed by a one- from the phylum Actinobacteria (60 isolates), including representatives way analysis of variance (ANOVA) with a least significant difference of Curtobacterium (50 of the 60 isolates, accounting for 83.33%), were (LSD) test, and the means were compared at a significance level of also major bacterial groups, but only 3 isolates were from the phylum P < 0.05. The program SPSS, version 17.0 (SPSS Inc., Chicago, IL, Bacteroidetes (Table 2). At the genus level, the most common bacterial USA) was used for statistical analysis. genera in the collection were Pantoea (31.25% of 608 isolates), Bacillus (25.99% of 608 isolates), and Pseudomonas (13.32% of 608 isolates) (Table S2 and Table S3). Species of the genus Curtobacterium (8.22% of 3. Results 608 isolates) have also been identified as frequently occurring en- dophytic bacteria in mulberry. Several genera, such as Sphingo- 3.1. Isolation of endophytic bacteria and bacterial community analysis bacterium, Klebsiella, and Streptomyces, only contained one isolate (Table 2). A total of 608 endophytic bacteria were isolated from all samples (127 from the winter community, 188 from the spring community, 293 from the autumn community, 147 from the CSQ community, 169 from

Table 2 Cumulative list of cultivable endophytic bacteria in mulberry and their taxonomic information.

Phyla Classes Orders Families Genera

Actinobacteria Actinobacteria Curtobacterium spp. (50) (60) Frigoribacterium spp. (2) Microbacterium spp. (4) Schumannella sp. (1) Geodermatophilales Geodermatophilaceae Geodermatophilus sp. (1) Corynebacteriales Nocardiaceae Rhodococcus sp. (1) Streptomycetales Streptomycetaceae Streptomyces sp. (1) Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae Agrobacterium spp. (5) (382) Ensifer sp. (1) Rhizobium spp. (15) Methylobacteriaceae Methylobacterium spp. (3) Brucellaceae Ochrobactrum sp. (1) Sphingomonadales Sphingomonadaceae Sphingomonas spp. (11) Sphingobium sp. (1) Novosphingobium spp. (2) Betaproteobacteria Burkholderiales Alcaligenaceae Achromobacter spp. (2) Comamonadaceae Comamonas sp. (1) Delftia spp. (3) Gammaproteobacteria Enterobacterales Enterobacteriaceae Atlantibacter spp. (19) Buttiauxella sp. (1) Cronobacter sp. (1) Enterobacter spp. (2) Klebsiella sp. (1) Kluyvera spp. (14) Leclercia spp. (2) Pantoea spp. (190) Erwiniaceae Erwinia spp. (7) Yersiniaceae Rouxiella sp. (1) Moraxellaceae Acinetobacter spp. (2) Pseudomonas spp. (81) Xanthomonadales Xanthomonadaceae Stenotrophomonas spp. (8) Xanthomonas spp. (8) Bacteroidetes Sphingobacteriia Sphingobacteriales Sphingobacteriaceae Sphingobacterium sp. (1) (3) Flavobacteriia Flavobacteriales Flavobacteriaceae Chryseobacterium spp. (2) Firmicutes Bacilli Bacillales Bacillaceae Bacillus spp. (158) (163) Paenibacillaceae Paenibacillus spp. (5)

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Fig. 1. Relative abundances (%) of cultivable endophytic bacteria in different communities isolated from mulberry at the phylum (A and B) and genus (C and D) levels. (A) and (C), communities isolated in different seasons, winter, spring, and autumn represent the community isolated in the winters, springs, and autumns of 2015 and 2016, respectively; (B) and (D), communities isolated from different mulberry cultivars, CSQ, CGS, HGE, and XLJ represent community isolated from Chuan Sang No. 7637, Changguo Sang, Hong Guo No. 2, and Xin Lunjiao, respectively.

Fig. 2. Venn diagram of endophytic bacteria isolated from mul- berry. (A) grouping by season, winter, spring, and autumn re- present the community isolated in the winters, springs, and au- tumns of 2015 and 2016, respectively; (B) grouping by cultivar, CSQ, CGS, HGE, and XLJ represent community isolated from Chuan Sang No. 7637, Changguo Sang, Hong Guo No. 2, and Xin Lunjiao, respectively. Values represent the number of genera.

3.2. Composition and diversity of endophytic bacteria in different community (34.33% of 67 isolates), whereas a higher abundance of communities Pantoea spp. in the spring community (56.52% of 92 isolates) and au- tumn community (74.72% of 178 isolates). Furthermore, parts of iso- Seasonal communities differed with respect to the richness of class lates were seasonally specific: Stenotrophomonas spp., Delftia spp., and and genus, although there was no obvious difference in bacterial Ensifer sp. were only detected in the winter community; Rhizobium spp. community variations at the phylum level: the winter community were not included in the spring community; and Paenibacillus spp. oc- harbored 4 phyla, 6 classes, and 18 genera; the spring community curred in the spring and autumn communities. Interestingly, noticeable harbored 4 phyla, 6 classes, and 22 genera; and the autumn community changes were not observed in the seasonal endophytes of the genera harbored 4 phyla, 5 classes, and 23 genera (Fig. 1A, C and Table S2). Bacillus and Curtobacterium, especially Bacillus spp. with a generally Gammaproteobacteria was the dominant class for each seasonal com- stable relative abundance (surpasses 23% in each season) (Fig. 1C, munity, accounting for 52.76, 48.94, and 60.75% of the total bacteria Table S2). isolated from the winter, spring, and autumn community, respectively. Meanwhile, the structures of the bacterial communities in mulberry However, within the class Gammaproteobacteria, seasonal variations were also influenced by the plant cultivars (Fig. 1B and D). At the resulted in a higher abundance of Pseudomonas spp. in the winter phylum level, Bacteroidetes occurred in all the mulberry cultivar

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are shown in the Venn diagram (Fig. 2). In all groupings, the number of shared genera (n = 10, Fig. 2A) among different seasonal communities was higher than that (n = 6, Fig. 2B) among different plant cultivar communities. Additionally, the number of shared genera was higher than the number of unique genera in a majority of the communities, except the CGS and CSQ communities. Among the shared genera, Pantoea, Bacillus, Pseudomonas, Curtobacterium, and Sphingomonas oc- curred in all communities, regardless of the season or cultivar com- munity, suggesting that the bacteria of these five genera are the core members of mulberry endophytes (Fig. 3). The biodiversity profiles of the 7 endophytic bacterial communities in mulberry were compared based on the diversity indices, including α- diversity indices (Shannon-Wiener index and Simpson’s diversity index) and Pielou’s evenness. For the seasonal communities, all the diversity indices for the winter community (H′ 2.18, D 0.83, E 0.75) were highest compared with that of the spring community (H′ 2.04, D 0.81, E 0.66) and autumn community (H′ 1.75, D 0.72, E 0.56) (Table 1). The di- versity of endophytic bacteria in mulberry markedly differed at dif- ferent sampling time, suggesting that season was an important factor for endophytic bacterial community variation. Regarding the mulberry cultivar communities, the Shannon-Wiener diversity index in the re- sistant cultivar communities (H′ 2.21 in CSQ and H′ 2.04 in CGS) was higher than that in the susceptible cultivar communities (H′ 1.67 in HGE and H′ 1.82 in XLJ). Moreover, the Simpson’s diversity index was highest in the CSQ community (D 0.84) and lowest in the HGE com- munity (D 0.70). Pielou’s evenness was mostly uniform in all the mul- berry cultivars and ranged between 0.62 and 0.71 (Table 1). The above observations indicated that resistant mulberry cultivars (CGS and CSQ) harbored more diverse endophytes than susceptible cultivars (XLJ and HGE).

3.3. Screening of antagonistic endophytes

A total of 100 isolates (16.45% of 608) exhibited antimicrobial activity against S. sclerotiorum, as revealed by the presence of an in- hibition zone (Fig. S2), and 68% of the antagonists belonged to the genus Bacillus (data not shown). Among the 100 antagonistic isolates, 33 strains showed stable and strong antifungal activity (Di > 1.0 cm). Fig. 3. Isolation frequency of the core mulberry endophytic bacteria in the The quantitative assay results also indicated that these strains exhibited communities isolated from different seasons (A) and different cultivars (B). varying degrees of antagonistic potential against the three target pa- Winter, spring, and autumn represent the community isolated in the winters, thogens, with inhibition rates ranging from 38.18% to 73.94% against springs, and autumns of 2015 and 2016, respectively; CSQ, CGS, HGE, and XLJ S. sclerotiorum, 33.20% to 69.50% against B. cinerea, and 19.44% to represent community isolated from Chuan Sang No. 7637, Changguo Sang, 72.21% against C. gloeosporioides (Table 3 and Fig. S2). Seven Bacillus Hong Guo No. 2, and Xin Lunjiao, respectively. strains (CW16-5, XW15-6, XS16-10, XS16-5, XA15-9, XP-27, and HA15- 19) efficiently antagonized S. sclerotiorum, and the inhibition rates were communities except the XLJ community, and the other three phyla higher than 68%, of which the isolate CW16-5 presented the highest (Actinobacteria, Firmicutes, and Proteobacteria) were detected in all antagonism (73.94%). Bacillus sp. XP-27, Pseudomonas sp. XA15-33, mulberry cultivar communities (Fig. 1B). At the genus level, the and Curtobacterium sp. XA15-35 exhibited outstanding inhibitory ac- number of genera in the communities of the resistant cultivars (22 tivity against all the tested phytopathogens, with inhibition rates genera in CGS and 23 genera in CSQ) was higher than that in the sus- greater than 51%, although the antagonistic activity of isolate XA15-35 ceptible cultivars (15 genera in HGE and 13 genera in XLJ) (Table 1). against C. gloeosporioides was slightly weak (40.61%) (Table 3). Additionally, although the isolates belonging to the genera Pantoea, The preliminary analysis results of antifungal mechanism showed Bacillus, Pseudomonas, Curtobacterium, Rhizobium, and Sphingomonas that at least one antibiotic biosynthesis-related gene could be amplified were present in all the mulberry cultivars, but the highest relative in all 33 target strains, and all positive amplification products were abundance of Bacillus spp. were detected in the CSQ community consistent with the expected size (Wang et al., 2016; Gond et al., 2015). (24.49%) and the HGE community (47.55%), respectively, whereas the Fourteen antagonists (42.42% of 33) had the potential to produce highest relative abundance of Pantoea spp. were detected in the CGS polyketides, and all 26 antagonistic Bacillus strains were found to community (40.83%) and the XLJ community (35.57%), respectively. harbor genes involved in lipopeptides biosynthesis (Table 3). The strain Among the isolates with low abundance, Agrobacterium spp. were only Bacillus sp. CW16-5 with marked antagonistic activity, tested positive detected in the CSQ and XLJ communities, Kluyvera spp. occurred in all for the genes PKSI, NRPS, sfp, srfC, and ItuD, suggesting that the ben- the mulberry cultivar communities except the CSQ community, and eficial effects of CW16-5 might be due to the direct antagonism toward Paenibacillus spp. were only observed in the CSQ and CGS communities phytopathogens through the efficient production of polyketide and two (Fig. 1D and Table S3). families of lipopeptides (surfactin and iturin). The common and unique bacterial genera in different communities

6 W. Xu, et al. Microbiological Research 229 (2019) 126328

Table 3 Determination of antimicrobial activity and functional genes associated with antibiotic biosynthesis for 33 antagonists.

No. Strain Inhibition rate ± SD (%) Functional genes

Ss a Bc a Cg a PKSI NRPS Sfp srfC ItuD FenD

1 Bacillus sp. CW15-1 50.55 ± 2.62 34.82 ± 1.27 43.59 ± 1.41 +b +-b +++ 2 Bacillus sp. CW15-2 57.15 ± 1.17 49.19 ± 2.23 29.03 ± 1.74 + + – +++ 3 Bacillus sp. CW15-3 56.36 ± 0.55 48.81 ± 1.65 31.10 ± 3.73 + + – +++ 4 Bacillus sp. CW15-4 50.97 ± 0.90 34.30 ± 1.26 36.31 ± 1.69 + + – +++ 5 Pantoea sp. CA15-44 39.21 ± 0.92 49.32 ± 1.35 44.25 ± 1.65 – ++––– 6 Pantoea sp. CA15-43 39.64 ± 0.55 57.77 ± 1.32 42.85 ± 0.38 + ––––– 7 Bacillus sp. CW16-5 73.94 ± 0.82 43.39 ± 1.46 37.39 ± 1.65 + + + + + – 8 Pantoea sp. CA15-30 38.18 ± 0.18 52.93 ± 0.59 38.88 ± 1.65 –– + ––– 9 Bacillus sp. XW15-3 58.00 ± 0.36 43.26 ± 1.24 56.24 ± 2.58 + + – +++ 10 Bacillus sp. XW15-6 70.67 ± 1.75 43.07 ± 0.73 50.79 ± 1.61 + + – ++– 11 Bacillus sp. XS16-9 50.24 ± 1.17 35.10 ± 0.39 31.18 ± 3.72 + + + + – + 12 Bacillus sp. XS16-10 71.64 ± 1.11 57.96 ± 1.37 51.03 ± 1.00 – +++–– 13 Bacillus sp. XS16-4 61.94 ± 0.64 50.55 ± 1.84 39.70 ± 3.02 + + + + – + 14 Bacillus sp. XS16-5 68.97 ± 0.38 57.58 ± 1.13 68.98 ± 1.49 + + – ++– 15 Bacillus sp. XA15-9 68.91 ± 0.36 48.48 ± 1.94 19.52 ± 4.09 – ++++– 16 Bacillus sp. XA15-10 62.18 ± 0.83 56.93 ± 1.18 21.92 ± 1.22 –– ++ + + 17 Curtobacterium sp. XA15-35 59.58 ± 1.94 55.64 ± 1.46 40.61 ± 2.25 –– ++ + – 18 Pantoea sp. XA15-46 56.48 ± 0.46 50.03 ± 2.07 26.14 ± 1.52 – ++++– 19 Pseudomonas sp. XA15-33 51.76 ± 1.18 52.10 ± 1.07 68.49 ± 2.21 + + –– – – 20 Bacillus sp. XA15-11 64.61 ± 0.86 49.97 ± 1.24 56.82 ± 1.14 – ++++– 21 Bacillus sp. XP-27 69.94 ± 1.00 69.50 ± 1.10 72.21 ± 2.23 + + – +++ 22 Bacillus sp. QW16-9 54.67 ± 1.91 59.77 ± 1.02 63.94 ± 1.83 – ++++– 23 Bacillus sp. QW16-12 54.67 ± 0.28 41.46 ± 0.87 59.39 ± 1.25 – +++++ 24 Bacillus sp. QW16-13 48.30 ± 1.11 61.64 ± 1.13 56.24 ± 1.17 – ++– + – 25 Rhizobium sp. QW16-15 57.27 ± 1.01 43.33 ± 1.18 19.44 ± 1.41 – +++++ 26 Bacillus sp. QW16-16 47.21 ± 0.46 49.13 ± 2.86 58.15 ± 2.24 – ++– + – 27 Bacillus sp. QW16-17 45.39 ± 0.47 46.42 ± 1.18 29.20 ± 2.11 – +++++ 28 Bacillus sp. HW16-12 59.45 ± 2.07 48.81 ± 1.46 57.65 ± 2.38 + + + + + – 29 Bacillus sp. HA15-7 51.45 ± 1.11 35.98 ± 1.51 54.51 ± 5.23 – +++–– 30 Bacillus sp. HA15-19 73.03 ± 1.51 33.20 ± 0.40 46.48 ± 2.65 – +++–– 31 Bacillus sp. HA15-23 50.36 ± 0.96 54.93 ± 0.58 52.44 ± 0.76 – ++– ++ 32 Bacillus sp. HA15-6 52.18 ± 0.83 50.93 ± 2.23 37.14 ± 1.45 – ++++– 33 Bacillus sp. HA15-34 55.88 ± 1.34 57.83 ± 1.35 67.00 ± 2.27 –– + –––

Percentage of fungal growth inhibition compared to the growth obtained in control plates. Data represent the mean ± standard deviation (SD) from three replicates. a (Ss) Sclerotinia sclerotiorum,(Bc) Botrytis cinerea,(Cg) Colletotrichum gloeosporioide. b (–) negative, (+) positive.

3.4. PGP traits and plant growth promotion assay of antagonistic mulberry seedling growth in comparison with the water-treated con- endophytes trol, as the shoot length of these seedlings increased by 75.06% and 21.48%, respectively, and the root fresh weight of these seedlings in- Among the 33 antagonists with stable and strong antifungal activ- creased by 158.06% and 103.88%, respectively. Furthermore, the strain ities, the isolates that tested positive for siderophore production up to Pantoea sp. CA15-44 increased both the root (43.64%) and shoot bio- 90.9%, followed by cellulase (78.8%), and protease production (72.7%) mass (206.14%) (Fig. 4). (Table 4 and Fig. S3). Twenty one isolates (63.6%) possessed nitrogen fixation activity, and phosphate solubilization was detected in 16 iso- lates (48.5%) (Table 4). In addition, the Pantoea spp. CA15-44 and 4. Discussion CA15-30 presented distinct phosphate solubilization as well as both siderophore production and nitrogen fixation. The isolates Bacillus spp. Plants harbor an abundance of endophyte, and some endophytic fi ff QW16-12 and HW16-12 could produce siderophores, cellulase, and bacteria exert several bene cial e ects on host plants, such as stimu- protease. This phenomenon revealed that mulberry endophytic an- lation of plant growth and enhancement of disease resistance (Hardoim tagonists also possess high PGP potential and might be good candidate et al., 2008; Reinhold-Hurek and Hurek, 2011; Porras-Alfaro and strains as biofertilizers. Bayman, 2011). The genetic diversity of endophytic bacteria is de- Eight strains with prominent antagonistic activity and high PGP termined by environmental conditions and host plant characteristics. potential, were selected from the dominant genera of endophytic bac- The former include climate, temperature, moisture, and drought stress, teria, and their effects on the growth of mulberry seedlings were further while the latter include various plant properties, such as genotype, evaluated in pot experiments (Fig. 4). The results showed that all the period of growth, and plant tissue (da Silva et al., 2014; Liotti et al., tested endophytes stimulated the growth of mulberry seedlings at dif- 2018; Mocali et al., 2003; van Overbeek and van Elsas, 2008). Eluci- fi ferent levels compared with the water-treated control, and the strain dation of the diversity of endophytic bacteria is bene cial for under- Bacillus sp. CW16-5 exhibited the highest PGP activities. When the standing the function and potential role of these bioactive bacteria in seedlings were treated with CW16-5, all the growth parameters, in- the micro-ecosystems of host plants. Currently, culture-dependent cluding root length, shoot length, and fresh weight of root and shoot, methods and culture-independent approaches have been successfully were significantly (P < 0.05) higher than those of the water-treated used for bacterial community analysis in diverse environments (Shen control, especially with shoot length and root fresh weight increasing and Fulthorpe, 2015; Mocali et al., 2003). With the development and by 83.37% and 217.70%, respectively. In addition, isolates Bacillus sp. implementation of next-generation sequencing (NGS) technologies, HW16-12 and Pseudomonas sp. XA15-33 also greatly facilitated culture-independent methods based on 16S rDNA amplicon sequencing in the Illumina MiSeq and HiSeq systems have played an important role

7 W. Xu, et al. Microbiological Research 229 (2019) 126328

Table 4 Characterization of antagonists for plant growth-promoting (PGP) traits and extracellular enzymes.

No. Strain PGP properties Extracellular enzymes

P-solubilization Siderophores Nitrogenase Celluase Chitinase Protease

1 Bacillus sp. CW15-1 – +++ – ++ – ++ 2 Bacillus sp. CW15-2 + +++ + ++ – ++ 3 Bacillus sp. CW15-3 + +++ – ++ + + 4 Bacillus sp. CW15-4 – +++ – +++ – +++ 5 Pantoea sp. CA15-44 +++ + + –– – 6 Pantoea sp. CA15-43 +++ + + –– – 7 Bacillus sp. CW16-5 +++ – + –– – 8 Pantoea sp. CA15-30 +++ + + –– – 9 Bacillus sp. XW15-3 – +++ – +++ – ++ 10 Bacillus sp. XW15-6 + +++ – +++ – ++ 11 Bacillus sp. XS16-9 – + – + – ++ 12 Bacillus sp. XS16-10 – ++ – ++ + + 13 Bacillus sp. XS16-4 + ++ + + + + 14 Bacillus sp. XS16-5 – +++ + ++ – + 15 Bacillus sp. XA15-9 – ++ + + – ++ 16 Bacillus sp. XA15-10 – ++ + ++ – +++ 17 Curtobacterium sp. XA15-35 +++ – + – ++ 18 Pantoea sp. XA15-46 – ++ + + –– 19 Pseudomonas sp. XA15-33 + – + – + – 20 Bacillus sp. XA15-11 ++ + – + – + 21 Bacillus sp. XP-27 – +++ – ++ – 22 Bacillus sp. QW16-9 + + + + – + 23 Bacillus sp. QW16-12 – ++ + ++ – + 24 Bacillus sp. QW16-13 – ++ – + – +++ 25 Rhizobium sp. QW16-15 – ++ + ++ –– 26 Bacillus sp. QW16-16 – ++ – +++ – +++ 27 Bacillus sp. QW16-17 + + –––+ 28 Bacillus sp. HW16-12 + +++ + ++ – + 29 Bacillus sp. HA15-7 + + + + – ++ 30 Bacillus sp. HA15-19 – +++– + 31 Bacillus sp. HA15-23 – ++ + + + – 32 Bacillus sp. HA15-6 + ++ + ++ – + 33 Bacillus sp. HA15-34 – ++ + + – +

For phosphate solubilization, + indicates diameters < 4 mm, ++ indicates diameters of 4∼8 mm, and +++ indicates diameters > 8 mm. For siderophore pro- duction, + indicates diameters < 2 mm, ++ indicates diameters of 2∼5 mm, and +++ indicates diameters > 5 mm. For extracellular enzyme activities (cellulase, chitinase, and protease), + indicates diameters < 5 mm, ++ indicates diameters of 5∼10 mm, and +++ indicates diameters > 10 mm. For nitrogenase activity, + indicates positive activity of the corresponding strain and – indicates no activity. Diameters represent the means of three replicates. in the analysis of complex microbial communities, but most information status of Bacillus species throughout the two years might be due to their regarding microbial diversity has been obtained using conventional inherent capacity with production of stable endospores, which persist cultivation techniques. for long periods in a number of microenvironments associated with In the present study, we adopted a culture-dependent approach to plants (Chen et al., 2014; Hu et al., 2014; Zhao et al., 2014). Ad- analyze seasonal fluctuations in the cultivable endophytic bacterial ditionally, it has been verified that different plant hosts can attract communities isolated from mulberry stems. To better understand the specific bacteria through the release of certain compounds via their relationship between microbial community and host cultivar, four roots, making these microorganisms successful endosphere colonizers healthy mulberry cultivars with different disease resistance properties (de Weert et al., 2002; Compant et al., 2005), and the Bacillus group were applied. A total of 608 bacterial isolates and 36 bacterial genera with strong environmental adaptability is a typical colonizer of various were collected from 24 mulberry samples (Table 2). In all analyzed crops (Compant et al., 2010; Liu et al., 2006). Bacillus strains were also samples, the genera Pantoea, Bacillus, and Pseudomonas were the major detected in all the mulberry cultivars used in our experiments, in other groups in the mulberry endophytic bacterial communities, and both the words, the presence of this group was independent of the mulberry host season and plant cultivar could affect the characteristics of these en- genotype. The data from this study revealed Bacillus was the pre- dophytic communities (Fig. 1 and Table 2). To the extent of our ponderant bacterial endophyte among all bacterial forms appearing in knowledge, this study is the first to characterize and compare the en- mulberry host, and it might be hinted in the importance of Bacillus dophytic bacterial communities in different mulberry cultivars over a genus for this plant genus. This speculation was consistent with the relatively long period. Although the diversity of endophytic bacteria functional description of mulberry endophytic B. subtilis 7PJ-16 in our was not investigated by the culture-independent technique, the culture- previous study, and strain 7PJ-16 was able to control mulberry fruit dependent method remains a valid tool for understanding the char- sclerotiniosis in the field and stimulate the mulberry seed germination acteristics of cultivable microorganisms and further obtaining func- as well as mulberry seedling growth under greenhouse conditions (Xu tional isolates with good bioactive capacities for use in plant protection et al., 2019). areas. Compared with chemical control, biological control through the use Notably, Bacillus was an extremely common mulberry endophytic of natural antagonistic microorganisms has emerged as a promising group in our study, regardless of season (Fig. 1A, C and Table S2) or strategy due to the rare environment contaminants and low health risks. cultivar communities (Fig. 1B, D and Table S3). This finding was con- As many literatures have verified, the development of new BCAs against sistent with observations for elm trees and other plants (Leifert et al., plant diseases requires the screening of high numbers of candidate 1989; Mocali et al., 2003; Shen and Fulthorpe, 2015). The dominant antagonists (Du et al., 2017; Liotti et al., 2018). Among these

8 W. Xu, et al. Microbiological Research 229 (2019) 126328

Fig. 4. Effects of the antagonists on the growth of mulberry seedlings under greenhouse conditions. (A) root and shoot lengths; (B) root and shoot fresh weights. Data are given as the mean ± SD of five replicates, and the different letters on the bars define groups of treatments that showed significant differences at the P < 0.05 level of confidence by one-way analysis of variance (ANOVA) and the least significant difference (LSD) test.

antagonists, members of the genus Bacillus (Chen et al., 2014; Hu et al., 2011; Santoyo et al., 2016). Our study concerning functional PGP traits 2014; Zhao et al., 2014; Sun et al., 2017a), Pseudomonas (Lee et al., and pot experiments found that many antagonists were positive for 2017; Sun et al., 2017b; Wicaksono et al., 2018), and Pantoea (Xie et al., several enzymatic activities and PGP traits, and some greatly stimulated 2017) can be considered ideal candidates. In recent years, Bacillus spp. the development of mulberry seedlings (Table 4 and Fig. 4). In parti- have received much attention because they are well-known antibiotic cular, Bacillus sp. CW16-5 showed the highest promotion activity in producers, and various Bacillus species have been found to control di- terms of all the growth parameters when compared with the water- verse phytopathogenic fungi and bacteria, such as Fusarium grami- treated control, including the root length, shoot length and fresh weight nearum (Dunlap et al., 2013; Zhao et al., 2014), S. sclerotiorum (Chen of the root and shoot. The roles and mechanisms of these mulberry et al., 2014; Hu et al., 2014; Sun et al., 2017a), and Xanthomonas oryzae endophytic bacteria with antifungal and PGP functions remain to be (Lin et al., 2001). In this study, 100 out of 608 strains were selected further characterized. from the mulberry endophytic bacterial community, of which 33 ex- Additionally, bacteria of the genus Curtobacterium, a high G + C hibited strong antagonistic activities against S. sclerotiorum (a pathogen gram-positive group, is worth mentioning because they are also that causes mulberry fruit sclerotiniosis). In addition, four strains of common endophytic inhabitants of mulberry (Fig. 3, Table S2, and Pantoea spp. (CA15-30, CA15-43, CA15-44, and XA15-46), one strain of Table S3). This observation was consistent with the presence of Curto- Pseudomonas spp. (XA15-33) and 26 isolates of Bacillus spp. also ex- bacterium species in many plants, including rice (Elbeltagy et al., 2000), hibited broad-spectrum antagonism toward different phytopathogens citrus (Araújo et al., 2001), and prairie plants (Zinniel et al., 2002). (Table 3). Using PCR assay, at least one functional gene associated with Several reports have indicated that Curtobacterium isolates can function antibiotic biosynthesis was detected in each antagonist. A majority of as biological control agents against many pathogens by inducing sys- the antagonists were positive for the presence of the lipopeptide genes, temic resistance (Raupach and Kloepper, 1998; Lacava et al., 2007)or including Sfp, srfC, ItuD, and FenD, and these microorganisms mainly producing antibiosis (Sturz and Matheson, 1996). Also, Sturz et al. originated from the group of Bacillus spp. with excellent antagonistic (1997) suggested that Curtobacterium luteum can stimulate intensive effects. Our study demonstrated that the antifungal effect of antag- plant growth for red clover (Trifolium pratense L.) when applied in- onistic Bacillus strains was closely associated with lipopeptide bio- dividually or in mixtures with Rhizobium strains. In our work, both cell synthesis. Similar results have been observed in numerous studies (Zhao suspensions and cell-free filtrates of Curtobacterium sp. XA15-35 ex- et al., 2014; Gond et al., 2015; Zouari et al., 2016). hibited obvious antifungal activity toward S. sclerotiorum (Table 3), and Apart from controlling diseases, endophytic bacteria have also at- the application of this strain had positive effects on the growth of tracted considerable attention for their capacity to promote plant mulberry seedlings (Fig. 4). The function of XA15-35 might be closely growth via direct or indirect mechanisms: 1) direct PGP mechanisms associated with the high phosphate solubilization as well as the pro- include nitrogen fixation, siderophore biosynthesis, phosphate solubi- duction of hydrolysis enzymes (chitinase and protease) (Table 4). lization, and phytohormone production such as indol-3-acetic acid Therefore, Curtobacterium sp. XA15-35 might also be a potential bio- (IAA), the former three of which are associated with plant nutrient logical resource for the biocontrol of mulberry fruit sclerotiniosis. acquisition; and 2) indirect PGP mechanisms include antifungal com- pound production and bioactive enzyme secretion, such as cellulases, 5. Conclusions chitinases, proteases, and these hydrolytic enzymes can promote the lysis of fungal cell walls (Jasim et al., 2013; Reinhold-Hurek and Hurek, The present research elucidated the structures, diversity, and

9 W. Xu, et al. Microbiological Research 229 (2019) 126328 functions of cultivable endophytic bacterial communities in mulberry. Seldin, L., 2014. Endophytic microbial community in two transgenic maize genotypes Overall, 608 isolates were obtained from all the mulberry samples, and and in their near-isogenic non-transgenic maize genotype. BMC Microbiol. 14, 332–341. these strains belonged to four bacterial phyla (Proteobacteria, de Weert, S., Vermeiren, H., Mulders, I.H.M., Kuiper, I., Hendrickx, N., Bloemberg, G.V., Firmicutes, Actinobacteria, and Bacteroidetes), including re- Vanderleyden, J., De Mot, R., Lugtenberg, B.J., 2002. Flagella-driven chemotaxis presentatives of Pantoea, Bacillus, Pseudomonas, Curtobacterium, and towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens. Mol. Plant Microbe Interact. 15 (11), 1173–1180. Sphingomonas. 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